Beta-galactosidase mutant, and preparation method and application thereof

ABSTRACT

The present invention discloses a β-galactosidase mutant, and a preparation method and application thereof, belonging to the fields of gene engineering and enzyme engineering. Amino acids of specific sites in the β-galactosidase are mutated, the β-galactosidase is transferred into a recombinant bacterium, and enzymatic transformation is performed under optimized conditions, so that the yield of galactooligosaccharide produced by the mutant reaches 59.8%, which is increased by about 70% as compared with that of wild enzyme, thereby implementing the increase of the galactooligosaccharide yield. The present invention has very high industrialized application value.

TECHNICAL FIELD

The present invention relates to a β-galactosidase mutant, and a preparation method and application thereof, belonging to the fields of gene engineering and enzyme engineering.

BACKGROUND

Galactooligosaccharide (GOS) is a functional oligosaccharide that is currently widely used in the food industry. As a new functional food additive, the GOS has attracted worldwide attention because of its unique physiological functions and excellent physicochemical properties. With the continuous advancing of development and research of GOS, coupled with abundant raw materials in China and the unlimited potential of the consumer market, the production of GOS has already set off a strong momentum all over the country. At present, the production of oligosaccharides in China is still an emerging industry. The development of GOS has not yet reached its scale. The main reason for restricting the production of GOS in China is the lack of industrial enzymes with excellent performance. Therefore, it is very important to find industrial enzymes with excellent performance.

β-galactosidase is a main enzyme used in industrial enzymatic method production of galactooligosaccharide. β-galactosidase derived from different microorganisms has different ability to generate GOS due to different properties. Currently known better GOS-producing strains are B. circulans, Kluyveromyces Lactis and A. oryzae. Although the yield of B. circulans (Bacillus circulans) is higher (48.3%), its product of GOS production is mainly 4′GalLac which has a poor probiotic effect. A main product of GOS produced by A. oryzae as a food-safe strain is 6′GalLac. 6′GalLac has a better probiotic effect than 4′GalLac, but its yield is only about 19%, which greatly limits its application.

Therefore, improving the yield of Aspergillus oryzae derived β-galactosidase for producing GOS to create conditions for its industrial production is a technical problem to be solved at present.

SUMMARY

The present invention firstly aims to provide a β-galactosidase mutant, wherein the mutant results from mutation of one or more amino acid sites of β-galactosidase of which an amino acid sequence is as shown in SEQ ID NO:2.

In an implementation of the present invention, the mutant results from mutation of one or more sites at the 140th, 264th, 304th and 806th positions of the β-galactosidase of which an amino acid sequence is as shown in SEQ ID NO:2.

In an implementation of the present invention, the mutant results from mutation of asparagine (Asn) at the 140th position of the β-galactosidase of which the amino acid sequence is as shown in SEQ ID NO:2 to cysteine (Cys), wherein the mutant is named N140C, the amino acid sequence is as shown in SEQ ID NO:13.

In an implementation of the present invention, the mutant results from mutation of phenylalanine (Phe) at the 264th position of the β-galactosidase of which the amino acid sequence is as shown in SEQ ID NO:2 to tryptophan (Trp), wherein the mutant is named F264W, the amino acid sequence is as shown in SEQ ID NO:14.

In an implementation of the present invention, the mutant results from mutation of phenylalanine (Phe) at the 304th position of the β-galactosidase of which the amino acid sequence is as shown in SEQ ID NO:2 to glutamine (Gln), wherein the mutant is named F3040.

In an implementation of the present invention, the mutant results from mutation of tryptophan (Trp) at the 806th position of the β-galactosidase of which the amino acid sequence is as shown in SEQ ID NO:2 to phenylalanine (Phe), wherein the mutant is named W806F.

In an implementation of the present invention, the mutant results from mutation of phenylalanine (Phe) at the 264th position of the β-galactosidase of which the amino acid sequence is as shown in SEQ ID NO:2 to tryptophan (Trp) and asparagine (Asn) at the 140th position to cysteine (Cys), wherein the mutant is named N140C/F264W.

In an implementation of the present invention, the mutant results from mutation of asparagine (Asn) at the 140th position of the β-galactosidase of which the amino acid sequence is as shown in SEQ ID NO:2 to cysteine (Cys) and tryptophan (Trp) at the 806th position to phenylalanine (Phe), wherein the mutant is named N140C/W806F.

The present invention secondly aims to provide a preparation method of the β-galactosidase mutant, which specifically comprises the following steps:

(1) according to determined mutant sites, designing mutagenic primers of site-directed mutagenesis, and performing site-directed mutagenesis by using a vector carrying the 3-galactosidase gene as a template; and constructing a plasmid vector containing the gene coding the mutant;

(2) transforming a mutant plasmid into a host cell; and

(3) selecting a positive clone, performing fermentation culture, and performing centrifuging, wherein supernate is a crude enzyme solution of the β-galactosidase mutant.

In an implementation of the present invention, the plasmid vector is any of pET series or pPIC9k.

The present invention thirdly aims to provide a method for preparing galactooligosaccharide by using the β-galactosidase mutant, which specifically comprises the following steps:

(1) by using lactose with concentration of 400 g/L-600 g/L as a substrate, adding the β-galactosidase, and adding an acetic acid-sodium acetate buffer, wherein pH is 4.5-5;

(2) performing enzymatic transformation in a shaking water bath, wherein reaction temperature is 40-60° C.;

(3) performing reaction within 5-36h, wherein a sample is taken every 4 hours during the reaction; and

(4) performing liquid-phase analysis on a reaction product, and calculating yield.

In an implementation of the present invention, an addition amount of the β-galactosidase is 2.5-5 U/mL.

The present invention has the following beneficial effects: by constructing the Aspergillus oryzae derived β-galactosidase mutant, the maximum GOS yield by transforming lactose is increased from 35.2% of a wild bacterium to 59.8%, so that the problem of lower industrial production yield of this enzyme is solved. Compared with the wild type, this enzyme has higher glucoside transformation activity, and is suitable for industrial production.

When the present invention is applied to galactooligosaccharide (GOS) production, optimum pH for production is 4.5, and optimum temperature is 40° C., which is more suitable for industrial production.

Based on the constructed mutant, the present invention optimizes the production of galactooligosaccharide (GOS) by enzymatic transformation, so that the industrial production value is higher. By performing enzymatic transformation under optimum conditions, the yield reaches 59.8%, which is increased by about 70% as compared with the wild type.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is optimum temperature for producing galactooligosaccharide by transforming lactose by using a wild enzyme and a mutant;

FIG. 2 is optimum pH for producing galactooligosaccharide by transforming lactose by using the wild type and the mutant.

DETAILED DESCRIPTION

BMGY liquid culture medium: YNB: 13.4 g/L; yeast extract: 10 g/L; peptone: 20 g/L; glycerol: 10 g/L; K₂HPO₄: 2.29 g/L; KH₂PO₄: 11.8 g/L; (NH₄)₂SO₄: 10 g/L;

BMMY liquid culture medium: YNB: 13.4 g/L; yeast extract: 10 g/L; peptone: 20 g/L; methanol: 40 g/L; K₂HPO₄: 2.29 g/L; KH₂PO₄: 11.8 g/L; (NH₄)₂SO₄: 10 g/L.

Enzyme Activity Measuring Method:

β-galactosidase activity analysis uses o-nitrophenyl-β-D-galactopyranoside (oNPG) as a substrate.

Prepare 20 mmol/L oNPG, take 100 μL of a properly diluted fermentation solution or enzyme solution and 1800 μL of 0.1 mol/L pH4.5 sodium acetate buffer, hold at the temperature of 60° C. for 5 min, adding 100 μL of the substrate, hold at the temperature of 60° C. for 10 min, immediately adding 1 mL of 1 mol/L precooled Na2CO3 solution to stop reaction and perform color development, and measure absorbance at 420 nm with a spectrophotometer.

Definition of enzyme activity unit: under the above analysis conditions, the amount of enzyme that catalytically produces 1 μmol of oNP per minute is one activity unit.

Analysis of Galactooligosaccharide (GOS) Yield by HPLC Method

A 60% (W/V) lactose solution is prepared as a substrate, and certain amounts of wild enzyme and double-mutant enzyme solutions are added, so that the final enzyme activity of the wild type enzyme solution is 10 U/mL and the final enzyme activity of the mutant is 2.5 U/mL (the effect of the amount of enzyme added on the production of GOS was previously performed, and the above conditions were respectively the optimum amounts of enzyme added for the mutant and the wild type). The reaction is performed under the optimum temperature and pH conditions, wherein the reaction for the wild type is performed for 10h (the time of the maximum yield of the wild type in a pre-experiment), and the reaction for the mutant is performed for 5h (the time of the maximum yield of the mutant in the pre-experiment). The reaction solution is centrifuged at 12000 rpm for 10 min, and supernate is taken, filtered through a 0.22 nm ultrafiltration membrane to remove 20 uL and subjected to HPLC analysis.

In the reaction solution, the amounts of disaccharides, trisaccharides, tetrasaccharides and pentasaccharides in a product are analyzed by HPLC. Chromatographic conditions for measurement are: Agilent 1200 HPLC chromatograph, Agilent automatic sampler, chromatographic column Hi-Plex Na, (300*7.7 mm), and differential detector Agilent G1362A; a mobile phase is pure water, a flow rate is 0.3 mL/min, and column temperature is set at 80° C.

Chromatographic conditions for measuring disaccharides are: Agilent 1200 HPLC chromatograph, Agilent automatic sampler, chromatographic column NH2-50 4E (4.6 mm*250 mm), and differential detector Agilent G1362A; a mobile phase is a 78% (v/v) acetonitrile-water mixed solution, a flow rate is 0.8 mL/min, and column temperature is set at 35° C.

Measuring Method of Galactooligosaccharide Yield: Yield (%)=(Mass of the galactooligosaccharide in product/Mass of all saccharides in product)×100%

The galactooligosaccharide yield is the sum of transferred disaccharides, transferred trisaccharides and transferred tetrasaccharides.

Example 1: Preparation of Single Mutant and Wild Enzyme

A β-galactosidase gene (SEQ ID NO:1) is connected to pMD19-T simple to obtain an AorE/pMD19-T simple plasmid, and the AorE/pMD19-T simple plasmid used as a template is subjected to mutation by using Sac1m-F and Sac1m-R as primers. An above PCR product is subjected to Dpn1 digestion, and the obtained plasmid is transformed into an Escherichia coli competent cell to obtain an AorE-M/pMD19-T simple plasmid.

SEQ ID NO: 3) Sac1m-F: TCCACAAGATCAGGGCTCTTGGTTTCAAC (mutant sites are underlined,  SEQ ID NO: 4) Sac1m-R: GTTGAAACCA AGAGCCCTGA TCTTGTGGA (mutant sites are underlined,  Preparation of wild enzyme: Snab1 and Not1 double-enzyme digestion is performed on the AorE-M/pMD19-T simple plasmid and a pPIC9k vector, enzyme digestion products are connected by a T4 ligase, a connection product is transformed into an Escherichia coli JM109 competent cell, and bacterium picking is performed to extract a plasmid, wherein the plasmid is named AorE-M/pPIC9k.

1. Preparation of recombinant bacterium Pichia pastoris: the AorE-M/pPIC9k plasmid is electrically transformed into a KM71 yeast competent cell, wherein the recombinant bacterium is named AorE-M/pPIC9k-KM71.

2. Preparation of single mutant: primers for N140C, F264W, F3040 and W806F mutations are respectively designed and synthesized, and the β-galactosidase gene is subjected to site-directed mutagenesis. The amino acid sequence of N140C is as shown in SEQ ID NO:13.

PCR amplification of site-directed mutant coding gene: by using a rapid PCR technique, PCR is performed by using a vector AorE-M/pMD19-T simple carrying a gene coding wild type maltooligosyl trehalose synthase as a template to respectively obtain mutated plasmids.

Mutagenic primers are as follows:

SEQ ID NO: 5) 140m-F: CCGGTTCGTACATCTGTGCCGAGGTCTCA (mutant sites are underlined,  SEQ ID NO: 6) 140m-R: TGAGACCTCGGCACAGATGTACGAACCGG (mutant sites are underlined,  SEQ ID NO: 7) 806m-F: CTCGACGAGAATTTCACGGTCGGCGAGGA (mutant sites are underlined,  SEQ ID NO: 8) 806m-R: TCCTCGCCGACCGTGAAATTCTCGTCGAG (mutant sites are underlined, SEQ ID NO: 9) 264m-F: AGCTATCCCCTCGGCTGGGATTGCGCAAACCCAT (mutant sites are underlined,  SEQ ID NO: 10) 264m-R: ATGGGTTTGCGCAATCCCAGCCGAGGGGATAGCT (mutant sites are underlined,  SEQ ID NO: 11) F304-Q: TCCAAGCGGGTGCTAATGACCCATGGGGTG (mutant sites are underlined., SEQ ID NO: 12) 264m-R: CACCCCATGGGTCATTAGCACCCGCTTGGA (mutant sites are underlined, 

Sequencing is respectively performed to confirm whether the coding gene of the β-galactosidase mutant is correct; Snab1 and Not1 double-enzyme digestion is performed on the plasmid carrying the mutant gene and the pPIC9k vector, enzyme digestion products are connected by a T4 ligase, a connection product is transformed into an Escherichia coli JM109 competent cell, bacterium picking is performed to extract the plasmid, and the plasmid is transformed into a yeast KM71 cell to obtain recombinant bacteria AorE-M/pPIC9k-N140C, AorE-M/pPIC9k-F264W, AorE-M/pPIC9k-F3040 and AorE-M/pPIC9k-W806F capable of expressing the mutant gene.

Example 2: Construction of Double Mutants

By using the plasmid of the mutant N140C constructed by the Example 1 as a template for double mutations, according to the primers of site-directed mutagenesis designed by the Example 1, site-directed mutagenesis is performed on the plasmid carrying the gene coding the mutant N140C by a rapid PCR technique to construct double mutants N140C/F264W and N140C/W806F. Sequencing is respectively performed to confirm whether a coding gene of the β-galactosidase double mutants is correct; Snab1 and Not1 double-enzyme digestion is performed on the plasmid with a correct sequencing result and the pPIC9k vector, enzyme digestion products are connected by a T4 ligase, a connection product is transformed into an Escherichia coli JM109 competent cell, bacterium picking is performed to extract the correct plasmid, and the plasmid is transformed into a yeast KM71 cell to obtain recombinant bacteria AorE-M/pPIC9k-N140C/F264W and AorE-M/pPIC9k-N140C/W806F capable of expressing the double mutant genes.

Example 3: Preparation of Wild Bacterium and Mutant β-Galactosidase Solutions

The recombinant bacteria AorE-M/pPIC9k/KM71, AorE-M/pPIC9k-N140C, AorE-M/pPIC9k-F264W, AorE-M/pPIC9k-F304Q, AorE-M/pPIC9k-W806F, AorE-M/pPIC9k-N140C/F264W and AorE-M/pPIC9k-N140C/W806F are respectively transferred into the BMGY liquid culture medium, and are cultured at 30° C. for 24h to obtain a bacteria solution. The bacteria solution is centrifuged, all thalli are transferred into 50 mL of the BMMY liquid culture medium, and cultured at 30° C. for 5 days, wherein methanol of which the final concentration is 0.75% is added every 24h to perform induced enzyme production. A fermentation solution is centrifuged, and supernate is a crude enzyme solution.

After the enzyme activity of the crude enzyme solution is measured, ONPG hydrolase activities of the wild type β-galactosidase (WT) and the mutant are listed in Table 1.

TABLE 1 Enzyme activities of wild type β-galactosidase and mutant enzymes Enzyme Enzyme Activity (U/mL) WT 185.3 N140C 9.7 F264W 23.2 F304Q 37.5 W806F 161.2 N140C/F264W 19.3 N140C/W806F 32.1

It can be seen from Table 1 that the mutants have lower oNPG hydrolase activity than the wild type. In this experiment, the enzyme activity and the yield are not directly proportional, and the level of the hydrolase activity does not represent the level of the yield. The hydrolase activity is only used as a quantitative measure of the amount of enzyme added.

Example 4: Optimum Temperature for Producing Galactooligosaccharide by Transforming Lactose by Using Mutant Enzyme

A 60% (W/V) lactose solution is prepared as a substrate, 60 g of lactose is dissolved in 50 mL of pH4.5 50 mml/L acetic acid buffer, and the volume of the solution is made up to 100 mL. Certain amounts of the wild enzyme and the double-mutant enzyme solution are added, so that the final enzyme activity of the enzyme solution is 2.5 U/mL; the enzyme solution is subjected to reaction at different temperatures, wherein the reaction for the wild type is performed for 10h, the reaction for the mutant is performed for 5h, and the reaction time is determined by the pre-experiment; and the galactooligosaccharide yield is analyzed.

It can be seen from FIG. 1 that the optimum enzymatic transformation temperature for the wild-type β-galactosidase is 60° C., the optimum enzymatic transformation temperature for the mutants is 40-60° C., and the maximum yield of the wild enzyme is 35.7% while the maximum yield of different mutants is 47.7% to 59.8%, which is greatly improved as compared with the wild enzyme. The maximum yield of the N140C/F264W double-mutant reach 59.8%, which is about 1.67 times that of the wild enzyme. In addition, as can be seen from the accompanying drawing, the temperature has little effect on the wild enzyme and the mutants at 40-60° C., so the present invention is more suitable for industrialized application.

Example 5: Optimum pH for Producing Galactooligosaccharide by Transforming Lactose by Using Mutant Enzyme

A 60% (W/V) lactose solution is prepared as a substrate, 60 g of lactose is dissolved in 50 mL of 50 mml/L acetic acid buffers with different pH, and each solution is made up to 100 mL. Certain amounts of the wild enzyme and the double-mutant enzyme solution are added, so that the final enzyme activity of the enzyme solution is 2.5 U/mL. The reaction temperature of the wild type is 60° C., and the reaction temperature of the mutants is determined according to the optimum temperature in the Example 4, ranging from 40 to 60° C. The reaction for the wild type is performed for 10h, and the reaction for the mutants is performed for 5h, wherein the reaction time is determined by the pre-experiment; and the galactooligosaccharide yield is analyzed.

It can be seen from FIG. 2 that except that the optimum pH of N140C is 5, the optimum pH of other mutants is 4.5. The maximum yield of the wild-type enzyme reaches 35.7% while the maximum yield of different mutants is 47.7%-59.8%, which is greatly improved as compared with controls. The maximum yield of N140C/F264W double-mutant reaches 59.8%, which is about 1.67 times that of the wild enzyme. 

What is claimed is:
 1. A gene coding a mutant β-galactosidase enzyme comprising a variant of the amino acid sequence of SEQ ID NO:2 with one or more mutations selected from N140C, F264W, F304Q, and W806F, wherein the enzyme catalyzes conversion of lactose to galactooligosaccharide (GOS).
 2. A vector or recombinant cell carrying the gene of claim
 1. 3. A method of making the mutant β-galactosidase enzyme using the recombinant cell of claim 2, comprising the following steps: performing a fermentation culture of the recombinant cell, and performing centrifuging to obtain a supernate from the fermentation culture, wherein the supernate is a crude enzyme solution comprising the mutant β-galactosidase enzyme. 